CN105315698B - A kind of synthesis and application of anthracene fluorochrome - Google Patents
A kind of synthesis and application of anthracene fluorochrome Download PDFInfo
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- CN105315698B CN105315698B CN201510155218.9A CN201510155218A CN105315698B CN 105315698 B CN105315698 B CN 105315698B CN 201510155218 A CN201510155218 A CN 201510155218A CN 105315698 B CN105315698 B CN 105315698B
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- substitution
- fluorescent dye
- alkyl
- substituted
- benzyl
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- 230000015572 biosynthetic process Effects 0.000 title abstract description 12
- 238000003786 synthesis reaction Methods 0.000 title abstract description 11
- MWPLVEDNUUSJAV-UHFFFAOYSA-N anthracene Chemical compound C1=CC=CC2=CC3=CC=CC=C3C=C21 MWPLVEDNUUSJAV-UHFFFAOYSA-N 0.000 title description 8
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 title description 5
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 26
- 125000005577 anthracene group Chemical group 0.000 claims abstract description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 41
- 238000006243 chemical reaction Methods 0.000 claims description 27
- 238000006467 substitution reaction Methods 0.000 claims description 27
- 239000003153 chemical reaction reagent Substances 0.000 claims description 21
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 17
- 229910052751 metal Inorganic materials 0.000 claims description 15
- 239000002184 metal Substances 0.000 claims description 15
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 13
- 125000003545 alkoxy group Chemical group 0.000 claims description 13
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 12
- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 claims description 11
- -1 alkane Epoxide Chemical class 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 10
- 230000005284 excitation Effects 0.000 claims description 9
- 239000001257 hydrogen Substances 0.000 claims description 9
- 229910052739 hydrogen Inorganic materials 0.000 claims description 9
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 9
- 150000001875 compounds Chemical class 0.000 claims description 8
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical class C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 claims description 8
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- 125000003118 aryl group Chemical group 0.000 claims description 7
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 claims description 7
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 7
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 6
- 239000000460 chlorine Substances 0.000 claims description 6
- 229910052801 chlorine Inorganic materials 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 claims description 5
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 5
- QILSFLSDHQAZET-UHFFFAOYSA-N diphenylmethanol Chemical class C=1C=CC=CC=1C(O)C1=CC=CC=C1 QILSFLSDHQAZET-UHFFFAOYSA-N 0.000 claims description 5
- 239000011737 fluorine Substances 0.000 claims description 5
- 229910052731 fluorine Inorganic materials 0.000 claims description 5
- UHOVQNZJYSORNB-UHFFFAOYSA-N monobenzene Natural products C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 5
- IAVREABSGIHHMO-UHFFFAOYSA-N 3-hydroxybenzaldehyde Chemical compound OC1=CC=CC(C=O)=C1 IAVREABSGIHHMO-UHFFFAOYSA-N 0.000 claims description 4
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical group OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 claims description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- 229910019213 POCl3 Inorganic materials 0.000 claims description 4
- 229940054021 anxiolytics diphenylmethane derivative Drugs 0.000 claims description 4
- RWCCWEUUXYIKHB-UHFFFAOYSA-N benzophenone Chemical compound C=1C=CC=CC=1C(=O)C1=CC=CC=C1 RWCCWEUUXYIKHB-UHFFFAOYSA-N 0.000 claims description 4
- 239000012965 benzophenone Substances 0.000 claims description 4
- 235000010290 biphenyl Nutrition 0.000 claims description 4
- 239000004305 biphenyl Substances 0.000 claims description 4
- 230000018044 dehydration Effects 0.000 claims description 4
- 238000006297 dehydration reaction Methods 0.000 claims description 4
- CZZYITDELCSZES-UHFFFAOYSA-N diphenylmethane Chemical class C=1C=CC=CC=1CC1=CC=CC=C1 CZZYITDELCSZES-UHFFFAOYSA-N 0.000 claims description 4
- CNTIXUGILVWVHR-UHFFFAOYSA-N diphosphoryl chloride Chemical compound ClP(Cl)(=O)OP(Cl)(Cl)=O CNTIXUGILVWVHR-UHFFFAOYSA-N 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- 125000004464 hydroxyphenyl group Chemical group 0.000 claims description 4
- 229910052744 lithium Inorganic materials 0.000 claims description 4
- WSFSSNUMVMOOMR-BJUDXGSMSA-N methanone Chemical class O=[11CH2] WSFSSNUMVMOOMR-BJUDXGSMSA-N 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 125000001424 substituent group Chemical group 0.000 claims description 4
- 125000003107 substituted aryl group Chemical group 0.000 claims description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 claims description 3
- 229910006069 SO3H Inorganic materials 0.000 claims description 3
- 125000001475 halogen functional group Chemical group 0.000 claims description 3
- 238000007363 ring formation reaction Methods 0.000 claims description 3
- 239000011701 zinc Substances 0.000 claims description 3
- 229910052725 zinc Inorganic materials 0.000 claims description 3
- 229930185605 Bisphenol Natural products 0.000 claims description 2
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 2
- 150000003935 benzaldehydes Chemical class 0.000 claims description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 2
- 229910052794 bromium Inorganic materials 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 2
- 125000001072 heteroaryl group Chemical group 0.000 claims description 2
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 2
- 229940098779 methanesulfonic acid Drugs 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 claims description 2
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 230000004044 response Effects 0.000 claims description 2
- WVLBCYQITXONBZ-UHFFFAOYSA-N trimethyl phosphate Chemical compound COP(=O)(OC)OC WVLBCYQITXONBZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims 7
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 claims 1
- 150000001555 benzenes Chemical class 0.000 claims 1
- 150000005204 hydroxybenzenes Chemical class 0.000 claims 1
- 238000006798 ring closing metathesis reaction Methods 0.000 claims 1
- 238000010189 synthetic method Methods 0.000 abstract description 15
- 230000004048 modification Effects 0.000 abstract description 11
- 238000012986 modification Methods 0.000 abstract description 11
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 abstract description 4
- OALHHIHQOFIMEF-UHFFFAOYSA-N 3',6'-dihydroxy-2',4',5',7'-tetraiodo-3h-spiro[2-benzofuran-1,9'-xanthene]-3-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 OALHHIHQOFIMEF-UHFFFAOYSA-N 0.000 abstract description 3
- 230000026731 phosphorylation Effects 0.000 abstract description 2
- 238000006366 phosphorylation reaction Methods 0.000 abstract description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 20
- 239000000975 dye Substances 0.000 description 15
- 239000000243 solution Substances 0.000 description 14
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 11
- 238000003756 stirring Methods 0.000 description 11
- 239000000047 product Substances 0.000 description 10
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 7
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 5
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- 229910052786 argon Inorganic materials 0.000 description 4
- ILAHWRKJUDSMFH-UHFFFAOYSA-N boron tribromide Chemical compound BrB(Br)Br ILAHWRKJUDSMFH-UHFFFAOYSA-N 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
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- 239000007787 solid Substances 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 241001251200 Agelas Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000007832 Na2SO4 Substances 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 3
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 238000006862 quantum yield reaction Methods 0.000 description 3
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- 229910052938 sodium sulfate Inorganic materials 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- PHDIJLFSKNMCMI-ITGJKDDRSA-N (3R,4S,5R,6R)-6-(hydroxymethyl)-4-(8-quinolin-6-yloxyoctoxy)oxane-2,3,5-triol Chemical compound OC[C@@H]1[C@H]([C@@H]([C@H](C(O1)O)O)OCCCCCCCCOC=1C=C2C=CC=NC2=CC=1)O PHDIJLFSKNMCMI-ITGJKDDRSA-N 0.000 description 2
- HIHOEGPXVVKJPP-JTQLQIEISA-N 5-fluoro-2-[[(1s)-1-(5-fluoropyridin-2-yl)ethyl]amino]-6-[(5-methyl-1h-pyrazol-3-yl)amino]pyridine-3-carbonitrile Chemical compound N([C@@H](C)C=1N=CC(F)=CC=1)C(C(=CC=1F)C#N)=NC=1NC=1C=C(C)NN=1 HIHOEGPXVVKJPP-JTQLQIEISA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
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- JWIPGAFCGUDKEY-UHFFFAOYSA-L O[Cr](Cl)(=O)=O.C1=CC=NC=C1 Chemical class O[Cr](Cl)(=O)=O.C1=CC=NC=C1 JWIPGAFCGUDKEY-UHFFFAOYSA-L 0.000 description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 2
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- QDLAGTHXVHQKRE-UHFFFAOYSA-N lichenxanthone Natural products COC1=CC(O)=C2C(=O)C3=C(C)C=C(OC)C=C3OC2=C1 QDLAGTHXVHQKRE-UHFFFAOYSA-N 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- UBJFKNSINUCEAL-UHFFFAOYSA-N lithium;2-methylpropane Chemical compound [Li+].C[C-](C)C UBJFKNSINUCEAL-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- LGRLWUINFJPLSH-UHFFFAOYSA-N methanide Chemical compound [CH3-] LGRLWUINFJPLSH-UHFFFAOYSA-N 0.000 description 1
- 230000001035 methylating effect Effects 0.000 description 1
- 230000004879 molecular function Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 229910052714 tellurium Inorganic materials 0.000 description 1
- PORWMNRCUJJQNO-UHFFFAOYSA-N tellurium atom Chemical compound [Te] PORWMNRCUJJQNO-UHFFFAOYSA-N 0.000 description 1
- XJDNKRIXUMDJCW-UHFFFAOYSA-J titanium tetrachloride Chemical compound Cl[Ti](Cl)(Cl)Cl XJDNKRIXUMDJCW-UHFFFAOYSA-J 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- AVBGNFCMKJOFIN-UHFFFAOYSA-N triethylammonium acetate Chemical compound CC(O)=O.CCN(CC)CC AVBGNFCMKJOFIN-UHFFFAOYSA-N 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 125000001834 xanthenyl group Chemical group C1=CC=CC=2OC3=CC=CC=C3C(C12)* 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention provides a class new fluorescent dye structure and its synthetic method, such anthracene structure fluorescent dye has similar in appearance to performances such as high absorptivity, the high quantum production rates of fluoresceins molecule, while there is exciting and launch wavelength with more red shift.The synthetic method simple economy that the present invention is provided, base group modification is carried out suitable for a large amount of synthesis and to molecular structure.The present invention is listed by the way that the quasi-molecule is carried out into phosphorylation modification, the phosphatase substrate molecule that there is a class fluorescence can produce property can be obtained, with extensive biological applications prospect.
Description
Technical field
Having the novel fluorescence dyestuff of anthracene structure the present invention relates to a class, there is provided synthetic method and phosphoric acid derivatization institute band
The photoluminescent property come changes.
Technical background
With the development of modern biotechnology, fluorescent dye compound as a kind of molecular function probe life science,
The every field such as chemical biology are widely used, as people's research molecular diagnostic techniques, immunoassay detection etc.
The favourable weapon of aspect.In numerous fluorescent dyes, there is dye to possess a kind of property, i.e. dyestuff in itself by certain
Chemical group modification after, the dye derivate is to change its own original photoluminescent property, be usually expressed as fluorescence effect
Rate declines to a great extent or disappeared, and using the analog derivative as substrate participate in enzyme reaction or other kinds of reaction it
Afterwards, its modification group comes off, so as to discharge original parent pigment structure, recovers its photoluminescent property again.We claim this kind of glimmering
Photoinitiator dye possesses fluorescence generation property (Fluorogenic dye), and the fluorescence molecule after deriving can produce molecule for fluorescence
(Fluorogenic molecules).Because its unique property so that this quasi-molecule is applied in many fields, and after
The appearance of continuous more applications that wait in expectation.But thering is any must be pointed out, although existing fluorescent dye compound is in whole spectrum
In the range of be distributed in the extensive region from ultraviolet band to near-infrared, but only sub-fraction fluorescent dye can be for entering
Row fluorescence can produce sex modification, including the Coumarins (coumarin) for being in ultraviolet region, the xanthene of blue region
Class (such as fluorescein fluorescein), the resorufin class (resorufin) and the indone class of red area of orange areas.At present
From the point of view of, in these fluorochromes, the dye structure that excitation wavelength falls at green Region (such as 545nm) is seldom, and chases after
The fluorescent dye of long excitation wavelength and cheap excitation source is asked to be paid close attention to always by people, because except economic factor, long wavelength can
To reduce the spontaneous background of biological tissue, and there are smaller light injury, deeper tissue permeability.
Fluorescent dye can be given birth to find the suitable fluorescence in this region, people are conventional using to fluorescein or Luo Dan
The means that bright class dyestuff is chemically modified, on the one hand because this two fluorochrome has outstanding extinction coefficient and quantum production
Rate, the also relatively convenient of the chemical synthesis involved by structure of modification in addition, the fluoresceins dyestuff containing xanthene structure is especially
So.On the other hand, base group modification (such as methylating or halo) can make launch wavelength red shift under normal circumstances, such as many chlorine
The fluorescein emission wavelength in generation can be with red shift 70nm or so, but also causes fluorescence quantum yield degradation simultaneously.To Luo Dan
Although the modification such as methylate of bright class is unlikely to have a strong impact on quantum yield, it is difficult to pass through again that the nitrogen-atoms methylated more, which is,
The means such as acylated synthesize fluorescence can be estranged sub.
Another effective method for changing launch wavelength is by the way that the bridging oxygen atom in oxa anthracenes structure is replaced
For other atoms, there are some researches show contain the elements such as selenium, carbon, silicon, tellurium as the dye matrix structure of bridge formation atom to show
Write launch wavelength (Arden-Jacob etc Spectrochim.Acta, the Part A 57,2271-2283 of red shift fluorogen；
Kolmakov, K.etc Eur.J.Org.Chem.2010,3593-3610；Kolmakov, K.etc
Photochem.Photobiol.Sci.11,522-532.).Method accordingly, for the research of green Region fluorescent dye, Luke
D.Lavis etc. has done good trial (ACS Chem.Biol.2013,8,1303-1310), and they instead of with tertiary carbon atom
Bridging oxygen atom in fluorescein, it was demonstrated that such dyestuff possesses outstanding optical property.But its synthesis is related to extraordinary part ginseng
With transition metal-catalyzed and unstable intermediate generation, be unfavorable for scale synthesis and derivatization.Find suitable synthesis
Approach is still necessary with the more new dye structures of synthesis.
The content of the invention：
The purpose of the present invention be for fluorescence under green excitation wavelength at present can natural disposition dyestuff species and synthetic method not
Foot, proposes a kind of synthetic method of anthracene fluorochrome, can effectively improve yield, increases the mobility of derivatization, reduction
Extensive synthesis cost, improve fluorescence can sub optical property estranged, it is strong to promote anthracene class fluorescein in terms of molecular labeling
Using.
In the first aspect of the present invention, there is provided a kind of fluorescein analog fluorescein that carbon bridge replaces one by one or anthracene class
Fluorescein, with long wavelength, fluorescence can natural disposition derivatization it is simple and easy to apply, the dyestuff of extinction coefficient and quantum yield comparable to fluorescein
Molecule, with having structure formula (1)：
The present invention provides a kind of fluorescent dye using anthracene structure as parent, it is characterised in that：With knot such as formula (1) Suo Shi
Structure, wherein R0It may be selected from-H, phosphate, substituted phosphate；R1, R5Can be independent selected from-H, fluorine, chlorine, aryl, substituted aryl,
C1-C6 alkyl, the C1-C6 alkyl of substitution, C1-C6 alkoxies, the C1-C6 alkoxies of substitution；R2-4, R6-8, R11-13Can be independent
Be selected from-H, fluorine, chlorine, bromine, aryl, substituted aryl, heteroaryl ,-CO2H、-CO2R、-SO3H、-SO3R、-CH2CO2H、-
CH2CO2R、-CH2SO3H、-CH2SO3R、-CH2NH2、-CH2NHR、-NO2, C1-C6 alkyl, substitution C1-C6 alkyl, C1-C6 alkane
Epoxide, the C1-C6 alkoxies of substitution, C1-C6 alkoxy aryls, the C1-C6 alkoxy aryls of substitution, phenyl, substituted-phenyl, connection
Phenyl, substituted biphenyl base, benzyl, substituted benzyl, benzoyl, substituted benzoyl, wherein R be selected from C1-C6 alkyl, substitution
C1-C6 alkyl, C1-C6 alkoxies, the C1-C6 alkoxies of substitution, C1-C6 alkoxy aryls, the C1-C6 alkoxy aromatics of substitution
Base, phenyl, substituted-phenyl, xenyl, substituted biphenyl base, benzyl, substituted benzyl, benzoyl, substituted benzoyl；R9、R10
Can be independent selected from C1-C6 alkyl, substitution C1-C6 alkyl, C1-C6 alkoxies, substitution C1-C6 alkoxies, C1-C6 alkane
Epoxide aryl, the C1-C6 alkoxy aryls of substitution, benzyl, substitution benzyl.
The above-mentioned purpose of the present invention is realized by following technical scheme：
(1) by the adjacent halo m-hydroxybenzaldehyde with hydroxy-protective group or the congener and band with other substituents
There is between hydroxy-protective group hydroxy phenyl metal reagent or reacted with hydroxy phenyl metal reagent between substituent, added
It is benzhydrol derivative into product.
(2) above-mentioned benzhydrol derivative is converted into benzophenone derivates through peroxidization.
(3) above-mentioned benzophenone derivates and metal reagent are reacted, obtains the diphenylmethane derivatives of dialkyl group substitution.
(4) above-mentioned dialkyl group diphenylmethane derivatives are generated into metal aryl by being reacted at low temperature with metal reagent
Compound.
(5) above-mentioned aryle and benzaldehyde derivative are reacted, generation addition compound product is benzyl alcohol derivative.
(6) above-mentioned benzyl alcohol derivative is generated into methanone derivatives through peroxidating
(7) above-mentioned methanone derivatives are obtained into bisphenol compound by removing phenolic hydroxyl protecting group group, passes through dehydration
Ring closure reaction obtains target fluorescent thing.
According to presently preferred technical scheme, R0For hydrogen, R1For methyl, R2-8、R11-13For hydrogen, R9、R10For first
Base.
According to presently preferred technical scheme, R0For H2PO3 -, R1For methyl, R2-8、R11-13For hydrogen, R9、R10
For methyl.
According to presently preferred technical scheme, R9Or R10Independent is selected from methyl, ethyl or propyl group.
According to the preferred technical scheme of the present invention, in synthetic method, hydroxy phenyl metal reagent can be with the middle of step (1)
For grignard reagent or lithium reagent.
According to the preferred technical scheme of the present invention, in synthetic method, metal reagent is grignard reagent or alkane in step (4)
Base lithium reagent.
According to the preferred technical scheme of the present invention, in synthetic method, the metal reagent in step 3 is zinc alkyl.
According to the preferred technical scheme of the present invention, in synthetic method,
Dehydration ring closure reaction is in step 7, by generating target fluorescent compound with the strong acid heating response such as methanesulfonic acid.
According to the technical scheme of present invention further optimization, R0For H2PO3 -.Its synthetic method can be by claim
Fluorescent chemicals described in 1 is dissolved in solvent, adds POCl3 or pyrophosphoryl chloride, and water is added after reaction a period of time and enters water-filling
Solution, gained Phosphorylated products pass through HPLC separating-purifyings.
According to the preferred technical scheme of the present invention, in synthetic method, the solvent is dichloromethane, tetrahydrofuran, second
At least one of nitrile, DMF, trimethyl phosphate.
According to the preferred technical scheme of the present invention, in synthetic method, fluorescent chemicals described in claim 1 is dissolved in molten
Agent, under the conditions of -10~10 DEG C of reaction temperature, adds POCl3 or pyrophosphoryl chloride.
The excitation wavelength scope for the fluorescent dye that the present invention is provided is 510-590nm, preferably 530-580nm；Launch light
Wave-length coverage is 550-650nm, preferably 550-650nm.
In this class fluorescent chemicals structure, R1 groups are characterized as hydrogen, fluorine, chlorine, C1-C6 alkyl, substitution alkyl, alkoxy
Etc. non-affine nuclearity functional group, the spirane structure that closure is formed between two parts aromatic ring agent structure up and down is on the one hand avoided,
So as to avoid in anthracene structure two phenolic hydroxy groups of formation, make follow-up to carry out fluorescence unlatching/closing type to phenolic hydroxyl group and repair
Only one of which phenolic hydroxyl group reaction site during decorations, is more beneficial for derivatization modification；On the other hand, fluorescent chemicals is reduced in itself
Polarity, so as to add the permeability of cell membrane of such dyestuff, be conducive to research research in terms of living cells imaging, sequencing and
Using.
The present invention provides a kind of new anthracene fluorochrome and its synthetic method.The fluorescent dye possesses 510-590nm's
Excitation wavelength range, 550-650nm launch wavelength scope, has filled up the deficiency of this area, can be effectively applied to living thin
The technical fields such as born of the same parents' imaging, sequencing, with highly important application value.
The invention provides a class new fluorescent dye structure and its synthetic method, such anthracene structure fluorescent dye has phase
The performances such as high absorptivity, the high quantum production rate of fluoresceins molecule are similar to, while having exciting with more red shift and launching
Wavelength.The synthetic method simple economy that the present invention is provided, base group modification is carried out suitable for a large amount of synthesis and to molecular structure.The present invention
List by the way that the quasi-molecule is carried out into phosphorylation modification, can obtaining a class, there is fluorescence can produce the phosphatase substrate of property
Molecule, with extensive biological applications prospect.
Brief description of the drawings
Fig. 1, the excitation and emission spectra of fluorescent chemicals 1
Fig. 2, fluorescence spectrum compares before and after phosphatase substrate molecule 2 is hydrolyzed through phosphatase (CIP)
Embodiment：
All waterless operations are carried out under argon gas protection, in the reactor by drying.Used chemistry is former
Material, reagent, solvent etc. are represented as that the analysis directly bought is pure or chemically pure reagent if do not illustrated.Dry solvent used
Non-aqueous processing conventionally carry out.
The instrument and method of product separation identification：Tlc silica gel is the types of GF254 60 (production of Merk companies), is used
254nm and 360nm double-wavelength ultraviolet lamps are detected；Column chromatography silica gel is Qingdao Haiyang 200-300 mesh silica gel；Nuclear magnetic resoance spectrum is used
Varian VXR-500 nmr determinations；Hydrogen spectrum, carbon spectrum are using TMS as internal standard；Reverse-phase chromatography uses Agela Cheetah-2
Middle pressure preparative chromatograph, Agela AQ C-18 prepare post separation.
Specific embodiment in the specific embodiment of the invention, is only further illustrating for the present invention, and not enough
Into the limiting factor of the present invention.
Embodiment one：
The synthesis of anthracene structure fluorescein " Beijing orange " (Peking Orange)：
In shown formula (1), R1=Me, R0=H, R2-8,11-13=H, synthetic route is as follows
Reagent and reaction condition：I) THF, -40 DEG C；Ii) DCM, PCC, diatomite；iii)ZnMe2, TiCl4, DCM-40 DEG C；
Iv) t-Buli, THF；O-tolualdehyde；V) DCM, PCC；vi)BBr3；MeSO3H
1. synthesize (the bromo- 5- methoxyphenyls of 2-) -3- methoxy benzyl alcohols 1a
In the 250ml for being equipped with constant pressure funnel dries round-bottomed bottle, the 80ml of m-methoxyphenyl magnesium chloride is done
Dry tetrahydrofuran solution is cooled to -40 DEG C, and the 20ml of 3- methoxyl groups o-bromobenzaldehye (10.8g) is dried into tetrahydrochysene under argon gas protection
Tetrahydrofuran solution is added dropwise in reaction bulb by constant pressure funnel, is kept for -40 DEG C stir 2-6 hours, is monitored by TLC anti-
Should, after stopping reaction after the disappearance of raw material 3- methoxyl groups o-bromobenzaldehye, add 20ml saturated aqueous ammonium chlorides and reaction is quenched.Instead
Answer the rotated evaporimeter of mixture to add 100ml dichloromethane in two times after removing most tetrahydrofurans, closed after point liquid
And organic phase, organic phase is by saturated common salt water washing, Na2SO4Dry, Rotary Evaporators use silica gel column chromatography, expansion after concentrating
Agent petrol ether/ethyl acetate~10/1, collects the product point of Rf~0.3, obtains 15g colourless liquids, yield 93%.
1H NMR(CDCl3, 500MHz)：δ 7.39 (d, J=10Hz, 1H, Ar-H), 7.23 (t, J=10Hz, 1H, Ar-H),
7.13 (d, J=5Hz, 1H, Ar-H), 6.96 (m, 1H, Ar-H), 6.81-6.79 (m, 1H, Ar-H), 6.96 (m, 1H, Ar-H),
(s, the 3H) of 6.70 (dd, J=10Hz, 5Hz, 1H, Ar-H), 6.08 (d, 1H, CH), 3.77 (s, 3H), 3.7513C NMR
(126MHz, CDCl3) δ 159.70,159.29,143.76,143.48,133.41,129.51,119.33,115.12,
114.01,113.11,113.05,112.80,74.63,55.49,55.23.HRMS：Calcd for C15H15BrO3Na(M+
Na), 345.0097.Found, m/z 345.0096.
2. synthesize (the bromo- 5- methoxyphenyls of 2-) -3- methoxy benzophenones 1b
(the bromo- 5- methoxyphenyls of 2-) -3- methoxy benzyl alcohols 1a (6.5g) is taken to be dissolved in 80ml dichloromethane, quickly stir
Mix down and add 10g pyridinium chloro-chromates (PCC), 2-5h is reacted at room temperature, TLC monitors the reaction after raw material disappears substantially
Stop, reaction solution is filtered by diatomite drainage short column, filtrate is collected after eluent methylene chloride, through silicon after Rotary Evaporators concentration
The product point of Rf~0.6 is collected in glue post column chromatography, solvent petrol ether/ethyl acetate~10/1, after concentration slightly yellow solid
6.1g, yield 95%.
1H NMR(CDCl3, 500MHz)：δ 7.50 (d, J=10Hz, 1H, Ar-H), 7.45 (m, 1H, Ar-H), 7.35 (d, J
=10Hz, 1H, Ar-H), 7.31-7.29 (m, 1H, Ar-H), 7.16-7.14 (m, 1H, Ar-H), 6.91-6.88 (m, 1H, Ar-
H), (s, the 3H) of 6.87 (d, J=5Hz, 1H, Ar-H), 3.85 (s, 3H), 3.7913C NMR (126MHz, CDCl3) δ 195.40,
159.92,158.74,141.48,137.29,133.93,129.61,123.52,120.47,117.36,114.18,113.76,
109.69,77.05,55.65,55.49.HRMS：Calcd for C15H14BrO3(M+H), 321.0121.Found, m/z
321.0120.
3. synthesize 1- bromo- 4- methoxyl groups -2- (2- (3- methoxyphenyls isopropyl)) benzene 1c
70ml dichloromethane is added in dry 250ml round-bottomed bottles, is cooled to -40 DEG C, TiCl is added4(9ml), under stirring
Zinc methide solution (1M toluene solution 80ml) is slowly added dropwise to, is stirred 15 minutes under the conditions of -40 DEG C under argon gas protection.Take
(the bromo- 5- methoxyphenyls of 2-) -3- methoxy benzophenones 1b (6.1g) is dissolved in 30ml dichloromethane, this solution is added dropwise to
State in solution, -40 DEG C of holding stirring reaction 3 hours is to slowly warm up to 0 DEG C and is further continued for reaction 5~10 hours afterwards, TLC monitorings
The reaction is until raw material stops after disappearing substantially.Poured under the brown reaction solution is stirred in trash ice and reaction is quenched, mixture warp
Dichloromethane is extracted, Na2SO4Dry, with silica gel column chromatography after Rotary Evaporators concentration, solvent petrol ether/ethyl acetate~
15/1, the product point of Rf~0.6 is collected, 5.5g colourless liquids, yield 87% is concentrated to give.
1H NMR(CDCl3, 500MHz)：δ 7.38 (d, J=10Hz, 1H, Ar-H), 7.21 (d, J=5Hz, 1H, Ar-H),
7.18-7.14 (m, 1H, Ar-H), 6.87-6.81 (m, 1H, Ar-H), 6.72-6.69 (m, 1H, Ar-H), 6.63 (dd, J=
10Hz, 5Hz, 1H, Ar-H), 3.82 (s, 3H), 3.74 (s, 3H), 1.73 (s, 6H)13C NMR (126MHz, CDCl3)δ
159.51,158.70,151.23,148.67,135.90,133.52,128.94,119.03,115.78,113.10,111.93,
109.87,55.38,55.14,44.88,30.16,13.29.HRMS：Calcd for C17H20BrO2(M+H),
335.0642.Found, m/z 335.0641.
4. synthesize (4- methoxyl groups -2- (2- (3- methoxyphenyls) isopropyl) phenyl) (2- tolyls) methanol 1d
Compound 1c (3g) is added in dry 100ml round-bottomed bottles, 30ml anhydrous tetrahydro furans is added and is dissolved,
It is cooled under argon gas protection after the hexane solution (9~10mmol) that tert-butyl lithium is added dropwise under -78 DEG C, stirring, completion of dropping and protects
Hold temperature to react 1 hour in the range of -78 DEG C~-60 DEG C, then o-tolualdehyde (11.5mmol) is dissolved in 10ml and dry four
Hydrogen furans, is slowly added into -78 DEG C of compound 1c reaction solution with syringe, makes it slowly freely heat up after completion of dropping
Reaction 2~6 hours.Add 10ml saturated ammonium chloride solutions and reaction is quenched.Rotated evaporimeter removes most tetrahydrofurans
After add 100ml dichloromethane extract three times, organic phase obtains crude Compound 1d, is directly used in down through drying after concentration
Single step reaction.
1H NMR(CDCl3, 500MHz)：δ 7.35 (d, J=10Hz, 1H, Ar-H), 7.22 (d, J=5Hz, 1H, Ar-H),
7.12-7.00 (m, 5H, Ar-H), 6.85-6.81 (m, 1H, Ar-H), 6.79-6.74 (m, 2H, Ar-H), 6.63 (dd, J=
10Hz, 5Hz, 1H, Ar-H), 5.74 (s, 1H) 3.86 (s, 3H), 3.69 (s, 3H), 2.01 (s, 3H), 1.71 (s, 3H), 1.51
(s, 6H)13C NMR (126MHz, CDCl3) δ 159.70,158.72,152.42,148.54,140.92,136.84,133.41,
131.99,130.50,129.53,127.66,127.19,125.24,119.14,118.34,113.70,112.63,110.51,
110.34,69.11,55.24,55.09,43.46,33.20,30.64,19.58.HRMS：Calcd for C25H29O3(M+H),
377.2038.Found, m/z 377.2042.
5. synthesize (4- hydroxyls -2- (2- (3- hydroxy phenyls) isopropyl) phenyl) (2- tolyls) ketone 1e
Previous step compound 1d is dissolved in 40ml dichloromethane, 4g pyridinium chloro-chromates are added under quick stirring
(PCC) 2-4h, is reacted at room temperature, and TLC monitors the reaction until raw material stops after disappearing substantially, and reaction solution passes through diatomite drainage
Short column is filtered, and filtrate is collected after eluent methylene chloride, is purified after Rotary Evaporators concentration through silicagel column column chromatography, solvent oil
Ether/ethyl acetate~7/1, collects the product point of Rf~0.5, after concentration slightly yellow solid 2.9g.
6. synthesising target compound 1 " Beijing orange " (Peking Orange)
Compound 1e (2.9g) is taken to be dissolved in 40ml dry methylene chlorides, ice-water bath cooling is lower to be added dropwise Boron tribromide (2-5eq),
Completion of dropping continues to react 2~5 hours.It is carefully added into 20ml frozen water after completion of the reaction to be quenched, mixture continues to stir 30 minutes,
Use saturation NaHCO3The aqueous solution adjusts PH to~7, adds the extraction of 150ml dichloromethane, and organic phase is washed through washing, saturated common salt
Wash, anhydrous Na2SO4After drying, rotated evaporimeter concentration, then by vacuum oil pump decompressing and extracting, obtain slightly yellow grease.
5ml pyrovinic acids are added into the grease, 80~100 DEG C are heated under stirring, keep the lower reaction of heating to stop for 1 hour.Will
Poured under reactant stirring in trash ice, the solid of precipitation is collected by filtration, then by washing, vacuum drying obtains target production
The crude product of thing 1 about 2.2g.Crude product passes through silica gel column chromatography separating-purifying, obtains 1.8g Orange red solids " Beijing orange " (Peking
Orange), yield 72%.Accompanying drawing 1 is the excitation and emission spectra figure of product 1.
1H NMR(CDCl3, 500MHz)：δ 7.32-7.29 (m, 1H, Ar-H), 7.24-7.18 (m, 2H, Ar-H), 7.02
(d, J=5Hz, 1H, Ar-H), 6.95 (s, 1H, Ar-H), 6.94 (s, 1H, Ar-H), 6.86 (d, J=10Hz, 1H, Ar-H),
(s, the 3H) of 6.50 (dd, J=10Hz, 5Hz, 1H, Ar-H), 1.94 (s, 3H), 15.2 (s, 3H), 1.4813C NMR (126MHz,
CDCl3) δ 174.51,157.24,155.28,136.62,135.96,130.24,129.10,128.84,125.72,122.37,
120.66,118.89,114.02,46.98,40.15,32.50,31.93,19.52.HRMS：Calcd for C23H19O2(M-
H), 327.1386.Found, m/z 327.1390.
On this basis, the compound for obtaining the formula (1) of other substituted radicals belongs to the routine techniques hand of chemical field
Section.
Embodiment two：
Elbs reaction structure fluorescence can produce acid phosphatase substrate molecule 2
300mg compounds 1 are added in dry reaction bottle, 15mL dried over anhydrous dichloromethane is added, stirred.It is outstanding to this
Proton sponge, stirring 10 minutes are added in turbid liquid until mixture dissolves.- 20~0 DEG C is cooled to, 250uL trichlorine oxygen is added dropwise to
Phosphorus, keeps 0.5~2h of low-temp reaction.30mL sodium phosphate buffers are added, continue to stir 1 hour.By resulting solution point liquid, aqueous phase
By concentration, and through C-18 reverse-phase chromatographic column separating-purifyings, separation condition：AQ C-18 prepare chromatographic column (Agela 40g), 0-
50% acetonitrile/TEAA buffer solution for gradient elution, flow velocity 20ml/min.Gained contains target product cut by concentration, and refrigeration is protected
Deposit standby.MS(ESI)：Calcd for C23H20O5P (M-H), 407.1.Found, m/z 407.1.Accompanying drawing 2 is phosphatase substrate
Fluorescence spectrum compares before and after molecule 2 is hydrolyzed through phosphatase (CIP)
Embodiments of the invention are only being explained further for the present invention, are not construed as limiting.In embodiment 1 and implementation
On the basis of example 2, those skilled in the art are readily available the fluorescence molecule of other substituents.
Claims (14)
1. a kind of fluorescent dye using anthracene structure as parent, it is characterised in that：With structure shown in below formula (1)
Wherein R0It may be selected from-H, phosphate, substituted phosphate；
R1, R5Can be independent selected from-H, fluorine, chlorine, aryl, substituted aryl, C1-C6 alkyl, substitution C1-C6 alkyl, C1-C6 alkane
Epoxide, the C1-C6 alkoxies of substitution；
R2-4, R6-8, R11-13Can be independent be selected from-H, fluorine, chlorine, bromine, aryl, substituted aryl, heteroaryl ,-CO2H、-CO2R、-
SO3H、-SO3R、-NO2, C1-C6 alkyl, substitution C1-C6 alkyl, C1-C6 alkoxies, C1-C6 alkoxy aryls, substitution
C1-C6 alkoxy aryls, phenyl, substituted-phenyl, xenyl, substituted biphenyl base, benzyl, substituted benzyl, benzoyl, substituted benzene
Formoxyl, wherein R are selected from C1-C6 alkyl, the C1-C6 alkyl of substitution, C1-C6 alkoxies, the C1-C6 alkoxies of substitution, C1-C6
Alkoxy aryl, the C1-C6 alkoxy aryls of substitution, phenyl, substituted-phenyl, xenyl, substituted biphenyl base, benzyl, substitution benzyl
Base, benzoyl, substituted benzoyl；
R9、R10Can be independent selected from C1-C6 alkyl, substitution C1-C6 alkyl, C1-C6 alkoxies, substitution C1-C6 alcoxyls
Base, C1-C6 alkoxy aryls, the C1-C6 alkoxy aryls of substitution, benzyl, substitution benzyl.
2. the fluorescent dye according to claim 1 using anthracene structure as parent, it is characterised in that
R0For-H2PO3。
3. a kind of preparation method of fluorescent dye as claimed in claim 1, comprises the following steps：
(1) by the adjacent halo m-hydroxybenzaldehyde with hydroxy-protective group and with hydroxy benzenes fund between hydroxy-protective group
Category reagent reacts with hydroxy phenyl metal reagent between substituent, obtains addition compound product i.e. benzhydrol derivative
(2) above-mentioned benzhydrol derivative is converted into benzophenone derivates through peroxidization
(3) above-mentioned benzophenone derivates and metal reagent are reacted, obtains the diphenylmethane derivatives of dialkyl group substitution
(4) above-mentioned dialkyl group diphenylmethane derivatives are generated into metal aryl chemical combination by being reacted at low temperature with metal reagent
Thing
(5) above-mentioned aryle and benzaldehyde derivative are reacted, generation addition compound product is benzyl alcohol derivative
(6) above-mentioned benzyl alcohol derivative is generated into methanone derivatives through peroxidating
(7) above-mentioned methanone derivatives are obtained bisphenol compound, passes through dehydration ring closure by removing phenolic hydroxyl protecting group group
Reaction obtains target fluorescent thing.
4. a kind of preparation method of fluorescent dye as claimed in claim 2, its preparation process is：
Fluorescent dye described in claim 1 is dissolved in solvent, POCl3 or pyrophosphoryl chloride is added, adds after reaction a period of time
Enter water to be hydrolyzed, gained Phosphorylated products pass through HPLC separating-purifyings.
5. the fluorescent dye according to claim 1 using anthracene structure as parent, it is characterised in that
R0For hydrogen, R1For methyl, R2-8、R11-13For hydrogen, R9、R10For methyl.
6. the fluorescent dye according to claim 1 using anthracene structure as parent, it is characterised in that
R0For-H2PO3, R1For methyl, R2-8、R11-13For hydrogen, R9、R10For methyl.
7. the fluorescent dye according to claim 1 using anthracene structure as parent, it is characterised in that
Its excitation wavelength scope is 510-590nm；Wavelength of transmitted light scope is 550-650nm.
8. the fluorescent dye according to claim 1 using anthracene structure as parent, it is characterised in that
R9Or R10Independent is selected from methyl, ethyl or propyl group.
9. preparation method according to claim 3, it is characterised in that
Hydroxy phenyl metal reagent is RMgBr or lithium reagent in the middle of the step (1).
10. preparation method according to claim 3, it is characterised in that
Metal reagent is RMgBr or alkyl lithium reagents in the step (4).
11. preparation method according to claim 4, it is characterised in that
The solvent is dichloromethane, tetrahydrofuran, acetonitrile, DMF, at least one of trimethyl phosphate.
12. preparation method according to claim 4, it is characterised in that
Fluorescent dye described in claim 1 is dissolved in solvent, under -10~10 DEG C of reaction condition, add POCl3 or
Pyrophosphoryl chloride.
13. method according to claim 3, it is characterised in that
Metal reagent in step (3) is zinc alkyl.
14. method according to claim 3, it is characterised in that
Dehydration ring closure reaction is in step (7), by generating target fluorescent compound with added methanesulfonic acid thermal response.
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